REVIEW Open Access The role of m A, m C ... - Molecular Cancer

REVIEW Open Access

The role of m6A, m5C and Ψ RNAmodifications in cancer: Novel therapeuticopportunitiesPaz Nombela1, Borja Miguel-López1 and Sandra Blanco1,2*

Abstract

RNA modifications have recently emerged as critical posttranscriptional regulators of gene expression programmes.Significant advances have been made in understanding the functional role of RNA modifications in regulatingcoding and non-coding RNA processing and function, which in turn thoroughly shape distinct gene expressionprogrammes. They affect diverse biological processes, and the correct deposition of many of these modifications isrequired for normal development. Alterations of their deposition are implicated in several diseases, includingcancer. In this Review, we focus on the occurrence of N6-methyladenosine (m6A), 5-methylcytosine (m5C) andpseudouridine (Ψ) in coding and non-coding RNAs and describe their physiopathological role in cancer. We willhighlight the latest insights into the mechanisms of how these posttranscriptional modifications influence tumourdevelopment, maintenance, and progression. Finally, we will summarize the latest advances on the development ofsmall molecule inhibitors that target specific writers or erasers to rewind the epitranscriptome of a cancer cell andtheir therapeutic potential.

Keywords: Cancer, Inhibitors, Anti-cancer therapy, Proliferation, Migration, Epitranscriptome, RNA modifications,N6-methyladenosine, m6A, 5-methylcytosine, m5C, Pseudouridine, Ψ

IntroductionThe epitranscriptome landscape is very complex, withmore than 170 different types of chemical modificationsof RNA described to date to decorate coding and non-coding RNAs (ncRNAs) [1]. Their occurrence has beenwell documented for over 50 years, however their func-tion remains still widely unknown [2]. Thus, whileknown since the emergence of molecular biology, RNAmodifications were only coined as the “epitranscriptome”in 2015. The study of the function of these modificationsis now emerging and has shown to have big implicationsin human pathologies [3, 4]. For example, the role of

6-methyladenosine (m6A), the most abundant and bettercharacterized internal modification in messenger RNA(mRNA), is to regulate embryonic stem cells and cancercells self-renewal and to favour survival upon heat shockor DNA damage [5–7]. In addition to the roles of m6Amodification in mRNAs, adenosine methylation is alsofound in non-coding RNAs, such as microRNAs (miR-NAs), long non-coding RNAs (lncRNAs), and circularRNAs (circRNAs) regulating their biogenesis and func-tion [8–15]. We now begin to appreciate the plethoraof molecular processes that are finely regulated byRNA modifications ranging from RNA metabolism,decay, splicing or translation, localization, stability,turnover, binding to RNA binding proteins (RBPs) orother RNAs, and thereby diversifying genetic informa-tion. Similar to epigenetics, groups of proteins havebeen identified that specifically “write” (catalyse the

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* Correspondence: [email protected] de Investigación del Cáncer and Instituto de Biología Molecular yCelular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC) -University of Salamanca, 37007 Salamanca, Spain2Instituto de Investigación Biomédica de Salamanca (IBSAL), HospitalUniversitario de Salamanca, 37007 Salamanca, Spain

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deposition of a specific modification), “erase” (catalysethe removal of a specific modification), and “read”(recognize and bind modified nucleotides) therebyaffecting the fate of RNA. Other modificationshave been recently documented in mRNA includingN6,2′-O-dimethyladenosine (m6Am), 5-methylcytosine(m5C), 5-hydroxymethylcytosine (hm5C), pseudouridine(Ψ), 1-methyladenosine (m1A) or 2'-O-ribose methylation,although their molecular functions remain still widelyunknown [5, 16].RNA modifications are also present in other regulatory

ncRNAs, in fact the most modified RNAs are transferRNA (tRNAs) and ribosomal RNAs (rRNAs) and theirmodifications shape protein synthesis efficiency andfidelity. More than 100 modifications have been describedfor tRNA, being the anticodon loop one hotspot of modifi-cations and play key roles in accurate and efficient decod-ing in translation [17]. In rRNA, most modificationscluster around functional sites including the decoding siteand the peptidyl transfer centre (PTC), suggesting theirfunctional relevance in regulating protein synthesis [18,19]. Studies in humans, yeast, and bacteria have shownthat dynamic deposition of these modifications in rRNAregulate cell growth, and drug and stress sensitivity byfine-tuning translation and is a very conserved mechanism[17, 20–22]. For instance, in yeast, flies, worms, andhumans, alterations of m5C levels in rRNA favours thetranslation of stress response-decoding transcripts inorder to increase survival [23, 24]. Occurrence of Ψ resi-dues increases in mRNA in yeast under starvation andheat shock [25–27]. And lack of 2′-O-ribose methylationsin rRNAs decreases efficient translation and affects growthand sensitivity to antibiotics [28]. Similarly, the overalllevels of tRNA modifications change to reprogram proteintranslation by changing codon usage [29–31].The deposition of RNA modifications is dynamic, and

thereby allows rapid cellular responses to environmentalsignals [16, 25, 31–34]. The ability to adapt to changingmicroenvironments such as that of stress or chemother-apeutic drugs is crucial to ensure survival of tumourcells, indicating that RNA modifications could play im-portant roles in cancer. Historically, cancer has beenconsidered fundamentally as a disease characterized bystepwise accumulation of genetic or epigenetic alter-ations of different oncogenes and tumour suppressorgenes. However, compelling evidence indicates thatepitranscriptomics could also play a fundamental role inthis pathology. Through its ability to modulate manyprocesses of RNA metabolism, dynamic RNA modifica-tions have been shown to be important emerging regula-tors in cancer [3, 33, 35–38]. Although RNAmodifications are not generally considered cancerdrivers, cumulative evidence shows that their aberrantexpression is functionally related to survival,

proliferation, self-renewal, differentiation, stress adapta-tion, invasion, and resistance to therapy, all of which arehallmarks of cancer [24, 33, 35, 37, 39–43]. For example,dynamic changes for multiple RNA modifications can beobserved in the urine of cancer patients [44]. Most strik-ing it has been the extraordinary enlargement of experi-mental evidence that implicates alterations in theexpression of m6A writers, erasers or readers are associ-ated with increased risk of obesity and diabetes, infertil-ity and with tumour-suppressive or tumour-promotingscenarios [3, 45]. Other RNA modifying enzymes havebeen found to be altered in cancer. For example, in anaggressive breast cancer cell lines, 2′-O methylation ap-peared to be hypermodified in rRNA and correlated withaltered translation [46]. Mutations in the rRNA pseudo-uridine synthase DKC1, cause X-linked dyskeratosis con-genita (X-DC) characterized by impaired translation,hematopoietic stem cells differentiation failure and in-creased cancer susceptibility [47]. Alterations in tRNAmodifications have been also reported in cancer includingm5C or 5-methoxycarbonylmethyluridine (mcm5U) andcorrelate with altered protein translation [33, 48–51]. Allthese studies show that aberrant RNA modifications con-tribute to proliferation, self-renewal, migration, stressadaptation and survival of cancer cells and suggest thattargeting aberrant posttranscriptional modifications incancer cells may hold promise as an efficient therapy fortumours [52].In this review we will discuss the molecular and cellu-

lar functions of RNA modifications in modulating geneexpression programmes, with a focus on their roles incancer. We further summarize here recent studies thatelucidate the therapeutic potential of targeting their ab-errant deposition in cancer. We will focus our reviewarticle on m6A, m5C and Ψ in coding and non-codingRNAs as notable examples due to the advances in ourunderstanding of the role of these epitranscriptomicmarks in cellular functions including proliferation, self-renewal, survival to stress or migration. In addition, ex-pression alterations or mutations in m6A, m5C and Ψdepositing machineries have been documented incancer.

6-methyladenosinem6A deposition in coding and non-coding RNAN6-methyladenosine (m6A), a well-known posttranscrip-tional modification first discovered in 1974 [53, 54], hasbeen regarded as the most frequent internal modificationfound in mRNA from viruses to mammals, but also oc-curs in small ncRNA and lncRNA in many eukaryoticspecies [11, 55]. Around 0.1–0.4% of all mRNA adeninesare methylated at position N6, representing approxi-mately 3–5 modifications per mRNA (Fig. 1) [56, 57].The recent advent of genome-wide m6A mapping of

Nombela et al. Molecular Cancer (2021) 20:18 of 30

polyadenylated RNAs has yielded unprecedented insightsinto the m6A-methylome landscape. Most methods forglobal m6A detection rely on immunoprecipitation ofmethylated RNAs using specific antibodies that recog-nise m6A [32, 58]. Subsequent improvements usingultraviolet crosslinking steps to bind the methylatedRNA to antibodies have allowed the identification ofm6A sites at single-nucleotide resolution [59, 60]. Thesemethods have revealed that this modification is enrichedat 3′ untranslated regions (3′UTRs), near stop codons,within long internal exons, in intergenic regions, introns,and at 5’UTRs (Fig. 1) [32, 41, 59–61].Deposition of m6A has been also reported in ncRNAs

such as miRNA, lncRNA and circRNA [9, 14, 62–65].Deposition in miRNA is enriched in primary miRNAs(pri-miRNA), but not in precursor miRNAs (pre-miRNA) and m6A marks are usually located in bothintergenic and intragenic pri-miRNAs that contain ca-nonical METTL3 motifs [9]. As for circRNAs, despitebeing derived from mRNA exons, m6A-modified cir-cRNAs are frequently derived from exons not methyl-ated in mRNAs [62]. LncRNAs are generally defined astranscripts longer than 200 nucleotides, and yet despitesharing several features with coding mRNAs such as be-ing 5′capped, spliced and, polyadenylated, m6A residuesin lncRNAs are distributed along the whole body of thetranscript and are more present in lncRNAs thatundergo alternative splicing [65].

m6A writersThe deposition of m6A occurs into nascent pre-mRNAsduring transcription and it is carried out in the nucleus by amulticomponent methyltransferase complex, that includesthe S-adenosyl methionine (SAM) binding proteinmethyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) heterodimeric catalytic core (Fig. 2a)

[66–68]. METTL3 catalyses the conversion of adenosineto m6A through its methyltransferase domain, whileMETTL14 is responsible for the recognition of RNAsubstrates, and therefore the whole METTL3-METTL14heterodimer is required for the methylation process [69, 70].In addition, the currently defined methyltransferase complexis also composed of adapters. The RNA-binding motif pro-tein 15 (RBM15) is one of these adapters and is responsiblefor the initial recruitment of the complex to its target site inthe mRNA. The regulatory proteins Wilms’ tumour1-associating protein (WTAP) and KIAA1429 (also knownas VIRMA) are responsible for the complex formation [71,72]. The recently characterized zinc finger CCCH domain-containing protein 13 (ZC3H13) has been found to act as abridge between the adaptor RBM15 and WTAP [73].miRNAs are methylated by the METTL3-METTL14-WTAP-RBM15/15B-KIAA1429 complex [9]. More recently,a new study has identified a single enzyme, METTL16, asanother active m6A methyltransferase in human cells [74].METTL16 has been shown to methylate mostly small nu-clear RNAs, a number of intronic sites in pre-mRNAs andin addition other ncRNAs (Fig. 2c) [15, 74, 75].

m6A erasersDeposition of N6-methyladenosine is reversible and relieson an orchestrated and dynamic network of specificmethyltransferases but also demethylases or “erasers”.Two 6-methyladenosine demethylases have been iden-tified, fat mass and obesity-associated protein (FTO)and AlkB homolog 5 (ALKBH5), which are membersof the nonheme Fe (II)/α-ketoglutarate-dependentdioxygenases (Figs. 1 and 2a) [76, 77]. FTO shows apreference for demethylating N6,2′O-dimethyladeno-sine (m6Am) but also demethylates m6A [78, 79].Furthermore, the AlkB homolog 3 (ALKBH3), anothermember of this family which preferentially acts on

Fig. 1 Schematic diagram of the location of m6A, m6Am, m5C and Ψ modifications on mRNA. Blue ribbon represents the mRNA with m7G-capand a poly(A) tail. ATG and STOP codons are indicated. The writers, erasers, readers and function are listed in the text box attached to eachmodification. Modifications: m6A, 6-methyladenosine; m6Am, N6,2ʹ-O-dimethyladenosine; m5C, 5-methylcytosine; Ψ, pseudouridine. Proteins:METTL, methyltransferase-like; FTO, fat mass and obesity-associated protein; PUS, pseudouridine synthase; NSUN, NOL1/NOP2/SUN domain familymember; ALyREF, Aly/REF Export Factor; ZCCHC4, zinc-finger CCHC domain-containing protein 4; ALKBH5, Alpha-Ketoglutarate-DependentDioxygenase AlkB Homolog 5; YTHDC, YTH domain-containing; YTHDF, YTH domain-containing family


m6A in tRNAs [80]. While we start to appreciate thecomplexity of the m6A methylation machinery, yet itremains to be fully understood how the methylationmachinery selectively and dynamically targets specificregions of the transcriptome.

m6A readersm6A methylation acts as a unique recognition elementfor reader binding proteins that drive the biochemicalprocesses that occurred to marked RNAs [81]. Some ofthe m6A readers contain a common RNA binding do-main, the YTH domain, which include the family of

YTH domain-containing proteins 1 and 2 (YTHDC1and YTHDC2, respectively) and the YTH domain familyproteins 1, 2 and 3 (YTHDF1, YTHDF2 and YTHDF3,respectively) [82, 83]. Subsequently, other readers havebeen discovered; eukaryotic translation initiation factor3 (eIF3), heterogeneous nuclear ribonucleoprotein(HNRNPC and HNRNPA2/B1), insulin-like growth factor(IGF2BP1, IGF2BP2, and IGF2BP3), proline-rich andcoiled-coil-containing protein 2A (PRRC2A), and the fra-gile X mental retardation protein FMRP, among otherprotein readers described to date (Figs. 1 and 2a) [8, 13,61, 84–87].

Fig. 2 Molecular mechanism of m6A deposition in RNA, biological function and implications in human cancer. a-c m6A RNA methylationlandscape in mRNA (a) and ncRNA (b & c) mediated by writers (blue balloons), including METTL3, METTL14, WTAP, RBM15B, KIAA1429,ZC3H13 and METTL16, erasers (pink balloons) FTO and ALKBH5 and reader proteins (yellow balloons) YTHDF1, YTHDF2, YTHDF3,YTHDC1, YTHDC2 and HNRNPC, HNRNPG, HNRNPA2, HNRNPB1, IGF2BPs and eIF3, and their role in mRNA metabolism. d-g Aberrantm6A deposition in mRNA and ncRNAs promotes or suppresses tumour progression through METTL3/METTL14 upregulation (d) ordownregulation (e) and FTO/ALKBH5 upregulation (f) or downregulation (g). Red arrows indicate induction. Blue arrows with flat endrepresent inhibition


Molecular function of m6A deposition in RNARole of m6A in mRNARegarding the biological function of m6A deposition, thisdependents on the protein reader that identifies andbinds the modified mRNA (Fig. 2a). For example, theYTHDC protein subtypes are found mainly in the nu-cleus, and specifically YTHDC1 has been described as analternative splicing factor of pre-mRNA [88]. YTHDC1facilitates mRNA export too by recruiting nuclear trans-port receptors or by binding to m6A methyltransferasescomplexed with TREX mRNA export complex andmodified mRNAs [89, 90]. In contrast, YTHDF proteinsubtypes are predominantly found in the cytoplasm andregulate the fate of cytoplasmic modified mRNAs. Forexample, YTHDF1 and YTHDF3 improve the efficiencyof mRNA translation [91, 92], and YTHDF2 facilitatesmRNA decay through the CC4-NOT deadenylase com-plex [93, 94]. YTHDF3 may accelerate mRNA decay toothrough interacting with YTHDF2 [92]. YTHDC2 pro-tein can exist both inside and outside the nucleus, beingable to selectively bind to m6A mark of ncRNA, yet thebiological consequence upon binding is unknown [11].In the cytosol, YTHDC2 affects the translation efficiencyand abundance of its target mRNAs [95]. While the evi-dence so far has shown that m6A deposition mediate di-verse effects through a complex network of readers, arecent study has revealed evidence for a unifying func-tional model of m6A readers, where m6A predominantlyinfluences mRNA degradation through the combined ac-tion of three YTHDF member readers [96].Apart from the YTHDC and YTHDF members, other

reader proteins can modulate translation or stability. Forinstance, eIF3 subunits have been reported to affect ca-nonical and non-canonical cap-dependent translation.This protein binds preferentially to m6A within the 5′UTR of mRNA leading to enhanced translation [61]. Inaddition, evidence shows that IGF2BP promotes the sta-bility and storage of their targeted mRNAs [87].It must be considered too that the methylation of ade-

nines causes alterations in the secondary structure ofRNAs, which in turn may alter their interaction withreader proteins [97]. m6A compared to A promotes thedestabilization of A/U pairings and alters the thermosta-bility of RNA duplexes [98]. This structural change hasbeen already correlated with an alteration in the inter-action between HNRNPC1 and HNRNPG with mRNA[13, 84]. Other members of the HNRNP family, HNRNPA2/B1 could bind directly to m6A to regulate alternativesplicing events and processing of precursor miRNAs [8].

Role of m6A in non-coding RNAsSimilar to mRNA, the presence of m6A on miRNA,lncRNA, and circRNA can regulate their binding tom6A-readers which in turn regulate their processing and

maturation, abundance, translation, stability, location,function, or degradation [8–12, 99–101], but also dy-namically regulate many physiological and pathologicalprocesses including tumourigenesis [102–104] (Fig. 2b).For example, the methylation of primary miRNAs (pri-miRNAs) by METTL3 increases their binding toHNRNPA2/B1, which interacts and enables micropro-cessor complex unit DGCR8 to target pri-miRNAs, pro-moting the initiation of miRNA biogenesis and theirmaturation into miRNAs [8, 9]. METTL14 can directlyrecruit DCGR8 on the m6A modified pri-miRNA encod-ing for miR-126a and consequently affecting the levels ofmiR-126a [12]. In addition, METTL14 has been foundto be associated with chromatin during transcriptionalelongation [105], which may suggest that METTL14may contribute to the co-transcriptional recruitment ofthe microprocessor complex on pri-miRNA transcripts.Therefore, alternations of m6A levels by methylases orerasers differential expression could result in significantchanges in the mature pool of miRNAs.As recently reported m6A deposition can alter lncRNA

stability. GAS5-AS1 transcript improves its stability bym6A when modulated by ALKBH5, however m6A canpromote GAS5 lncRNA degradation through YTHDF2and YTHDF3 binding [99, 100]. Although it is unclearwhether lncRNA localization is also regulated by m6A, ithas been shown that overexpression of METTL3 cansignificantly increase the nuclear localization of RP11lncRNA [101]. The modification of m6A in lncRNAcould as well affect their structure and influence the in-teractions between RNAs and between RNAs and theproteins that regulate their specific biological functions[11, 13, 14]. For instance, HNRNPC was found to bindto the m6A-modified hairpin compared to the unmethy-lated hairpin of the lncRNA MALAT1 in an “m6Aswitch”-regulated manner, which indicated that m6Amodification disrupts lncRNA hairpin-stem structureand thus promoting its binding to HNRNPC [13].LncRNA X-inactive-specific transcript (XIST) is methyl-ated by METTL3, and METTL3 knockdown was shownto impair XIST-mediated transcriptional silencing ofgenes on the X chromosome both in vitro and in vivo[11]. Cytoplasmic lncRNA linc1281 is methylated at its3′-end region, and the methylation marks are requiredfor the binding of let-7 through the interaction of yetunknown proteins [14].Regarding METTL16-mediated deposition in snRNAs,

initial studies have shown to methylate A43 position of U6snRNA, which is found near the region that base pairs with5′ splice sites of pre-mRNAs, suggesting that METTL16plays an important role in mRNA splicing [15].Interestingly, ncRNAs also play significant roles in regu-

lating the methylation levels of adenosine-6 [106, 107].For example, in differentiating stem cells, miRNAs would


regulate the binding of METTL3 to mRNAs, by using asequence pairing mechanism, and thus modulating theabundance of m6A [107]. Also in hepatocellularcarcinoma (HCC), the inhibition of miR-145 causes anincrease in YTHDF2 expression which in turn leads to afall in the levels of m6A, probably due to increased mRNAdegradation [64].While the functions of m6A deposition are currently

not fully understood, a picture begins to emerge andshows that m6A methylation takes part in the regulationof mRNA processing, decay, stability, splicing, polyade-nylation, nuclear export and translation. In the case ofncRNAs, m6A has been shown to promote their process-ing or enhance their functions [8, 11].

The role of m6A in cancerNumerous studies have shown that m6A deposition inRNA plays a critical role in many physiological processes,including circadian rhythms regulation, spermatogenesis,embryogenesis, DNA damage and stress response, pluri-potency and cell reprogramming [7, 36, 41, 75, 108–110].Furthermore, emerging evidence suggests that m6A regu-lators are also closely associated with oncogenic ortumour suppressive functions including proliferation[111], tumourigenesis [112], invasion [113], metastasis[12, 114, 115] or immune system evasion [116] inmalignant tumours. Below, we summarize the mainemerging roles of m6A writers, erasers and readers incancer (Table 1).

Role of m6A writers in cancerMETTL3 and METTL14 expression have been reportedto promote tumourigenesis in several cancer types (Fig.2d). For example, METTL3 is overexpressed in acutemyeloid leukaemia (AML) [117]. In this study, METTL3was shown to methylate BCL-2, PTEN, and c-MycmRNAs which resulted in their increased translation,inhibiting cell differentiation and apoptosis, and promo-tion leukaemia progression [117]. However, the mechan-ism of the m6A-dependent translation remainsundetermined. Furthermore, another study identifiedthat METTL3 expression in vivo was essential to main-tain AML cells in an undifferentiated state, and thusmaintaining myeloid leukaemia growth [118]. In com-parison, METTL14 was shown to act as an oncogene inAML by increasing MYB and c-Myc mRNA stability andtranslation [40]. METTL3 acts as an oncogene that facil-itates the growth, survival, and invasion of cells in lungand colon cancer by promoting the translation of EGFRand TAZ [113]. METTL3 is also upregulated in breastcancer, where it increases methylation and stability ofHBXIP mRNA, which induces cell proliferation and sur-vival of tumour cells via inhibiting the tumour suppres-sor let-7 g [119]. Recent studies have shown that

aberrant overexpression of m6A modifiers is observed incolorectal cancer too. The aberrant deposition of m6Aincreased miRNA-1246 expression, resulting in thedownregulation of the tumour suppressor of SPRED2and metastasis induction [120]. Other recent studieshave reported that METTL3 is overexpressed in prostatecancer cells, contributing to the growth and invasion ofcancer cells through SHH-GLI1 signalling [121]. Inaddition, METTL3 also regulates the expression ofITGB1, thus affecting its binding to Collagen I, the mo-bility of tumour cells, and promoting prostate cancerbone metastasis [122].Despite the unanimously oncogenic functions of

METTL3 and METTL14 in all these cancer types, inglioblastoma and HCC several reports have demon-strated both, oncogenic and tumour suppressive roles.Initial studies showed that m6A methylation inhibitedthe growth, self-renewal, and tumourigenesis of glio-blastoma stem cells by decreasing the stability and ex-pression of key oncogenic transcripts such as ADAM19[35]. In contrast, in a subsequent study, METTL3 wasfound to be upregulated, and to be a predictor of poorpatient survival. In this study, METTL3 was found to in-crease the stability and expression of SOX2 mRNA, pro-moted the growth of glioblastoma stem cells (GSCs),and prolonged survival in mice [123]. Similarly, in HCCthe expression of m6A writes can inversely promote orsuppress tumourigenesis. For instance, METTL14 wasinitially identified as a tumour suppressor in HCC [12].In this study, the authors showed that METTL14 expres-sion induced an increase in pri-miR-126 expression, sup-pressing tumour metastases [12]. Conversely, in anotherstudy METTL3 was found upregulated and associatedwith poor prognosis in patients with HCC. In anotherstudy, METTL3 inhibited SOCS2 mRNA expressionthrough a m6A-YTHDF2 dependent manner [56]. Fromthe reported data one can conclude that the variable ex-pression of m6A writers and their bound factors, erasersand readers may account for differences in m6A depos-ition levels, as well as differences in targeted RNAs,which in turn will lead to the observed discrepancy andcontroversy. Yet more studies will be necessary to pre-cisely determine the nature of METTL3 and METTL14in glioblastoma and HCC and to identify the factors,pathways or tumour cell states responsible for the con-troversial findings.In other tumours, METTL3 and METTL14 play an

tumour suppressive role (Fig. 2e). For example, in endo-metrial cancer, 70% of tumours exhibit a reduction inm6A levels, either through METTL14 mutations ordownregulation of METTL3 expression. METTL3-METTL14 complex loss leads to increased cell prolifera-tion by upregulating the expression of members of theAKT pathway, and thus, showing a tumour suppressor


Table 1 Alterations in N6-methyladenosine writers, erasers and readers in cancer. AML: Acute myeloid leukaemia; HCC:Hepatocellular Carcinoma; GSC: Glioma Stem Cells; LncRNA: long non-coding RNA

Factor/Enzyme Cancer type Alteration Mechanism Ref

Writers

METTL3 AML Upregulated METTL3 promotes translation of oncogenes’ mRNAs, inhibiting celldifferentiation and apoptosis.

[117, 118]

Bladder Cancer Upregulated METTL3 accelerates miR221/222 maturation. [104, 157,321, 322]

Breast Cancer Upregulated METTL3 inhibits miRNA let-7 and induces proliferation and survival. [119]

Colon Cancer Upregulated METTL3 increases miRNA-1246 expression inducing metastasis. [120]

Endometrial Cancer Downregulated METTL3 regulates the expression of members of AKT pathway and inhibitscell proliferation.

[111]

Glioblastoma Downregulated METTL3 decreases oncogene expression and inhibits the self-renewal. [35, 136]

Glioblastoma/Gastric Cancer

Upregulated METTL3 increases the stability of oncogenic mRNAs. [123, 323]

HCC Upregulated METTL3 inhibits mRNA expression of tumour suppressors, increasingproliferation and metastasis.

[56, 114]

Lung/Colon Cancer Upregulated METTL3 promotes oncogenes’ mRNA translation. [113]

Lung Cancer Upregulated METTL3 increases miR-143-3p expression and induces oncogenes translation. [43, 324, 325]

Melanoma Upregulated Unknown. [326, 327]

Osteosarcoma Upregulated METTL3 increases mRNA expression of oncogenes. [328]

Ovarian Cancer Upregulated METTL3 stimulates the translation of oncogenes. [329, 330]

Prostate Cancer Upregulated METTL3 increases oncogene mRNA expression. [121]

Prostate Cancer Upregulated METTL3 increases the mRNA stability of cell adhesion genes. [122]

Renal CellCarcinoma

Upregulated Unknown. [124, 331,332]

METTL14 AML Upregulated METTL14 increases oncogenes’ mRNA stability and translation. [40]

Bladder Cancer Downregulated METTL14 increases oncogenes’ mRNA decay. [333]

Colon Cancer Downregulated METTL14 regulating primary miRNA processing of YAP an SP1 pathways ordownregulating lncRNA XIST.

[334, 335]

EndometrialCancer

Downregulated METTL14 regulates the expression of members of AKT pathway and inhibitscell proliferation.

[111]

HCC Downregulated METTL14 induces an increase of pri-miR-126 expression that suppressestumour metastases.

[12]

Renal CellCarcinoma

Downregulated METTL14 represses the translation of P2RX6 increasing invasion. [336]

Pancreatic Cancer Downregulated Unknown. [337]

Erasers

FTO AML Upregulated FTO enhances oncogenes’ mRNA stability. [36, 37]

Bladder Cancer Downregulated Unknown. [338]

Breast Cancer Upregulated FTO downregulates the expression of tumour suppressor genes. [134]

Cervical Cancer Upregulated FTO promotes translation of oncogenes, promoting migration and drugresistance.

[131, 132]

Glioblastoma No change FTO regulates of oncogene expression. [35, 136]

HCC Downregulated Unknown. [339]

Lung Cancer Upregulated FTO regulates MZF1 expression. [133]

Melanoma Upregulated FTO increases stability of critical immunotherapy resistance and pro-tumorigenic melanoma cell-intrinsic genes.

[38, 130]

Pancreatic Cancer Upregulated FTO promotes oncogene mRNA stability. [340]

ALKBH5 AML Upregulated Unknown. [341, 342]

Breast Cancer Upregulated ALKBH5 enhances mRNA stability of stem cell self-renewal genes. [135, 137]


function in endometrial cancer [111]. Similarly, in renalcell carcinoma, depletion of METTL3 promotes cellproliferation, growth, and colony formation through thePI3K-AKT-mTOR pathway activation and enhances cellmigration and invasion through the epithelial-mesenchymal transition (EMT) pathway [124].The implication of METTL16 in cancer is poorly under-

stood, in fact only few studies have linked METTL16 tocancer [15, 125]. In recent studies, loss-of-function muta-tions and expression alterations were found in colon can-cer, suggesting a role for METTL16 in the tumourigenesisof colorectal cancer [126, 127]. Other studies have associ-ated METTL16 with the maturation of MALAT1 mRNAwhich can act as an oncogene and a tumour suppressor indifferent types of cancer [125]. In addition, given the roleof METTL16 in regulating snRNA methylation and hencetheir processing, METTL16 dysregulation could favourtumour development by inducing changes in alternativesplicing. Studies in the last decade have demonstrated thepotential role of alternative splicing in the aetiology ofcancer [128]. Indeed, the change in the expression of keyenzyme isoforms in apoptosis, metabolism, cell signallingand resistance to therapy has been attributed to theacquisition of the tumour phenotype too [128].

Role of m6A erasers in cancerDysregulation of m6A erasers have been found too asso-ciated to cancer risk, in fact FTO polymorphisms havebeen known to be associated to several human disordersincluding increased risk of cancer for decades [129].After the discovery of FTO catalytic activity, we begin to

understand the molecular mechanisms underlying FTOoncogenic activity. Initial studies showed that FTO ishighly expressed in AML and exerts its oncogenicfunction by reducing m6A levels in the mRNA of thetumour suppressors RARA and ASB2, leading to inhib-ition of their expression [36]. Similarly, FTO was shownto induce GSCs growth, self-renewal, tumour progres-sion, and was associated with poor survival throughregulation of ADAM19 (Fig. 2f) [35]. More recentstudies have linked increased expression of FTO to othertumours. For example in melanoma, FTO expressionpromotes tumourigenesis and resistance to immuno-therapy (anti-PD-1) by directly removing m6A fromPDCD1, CXCR4, and SOX10 mRNAs, thereby increas-ing their stability (Fig. 2f) [38, 130]. FTO is also up-regulated in cervical cancer where it inducesresistance to chemo-radiotherapy and enhances theresponse to DNA damage [131, 132]. In cervical can-cer, FTO can activate the β-catenin pathway and in-crease ERCC1 expression that is associated withworse prognosis. Furthermore, FTO promotes cell mi-gration and proliferation by positive regulation ofE2F1 and MYC [131, 132]. In lung cancer FTO isfound overexpressed and is associated with poorerprognosis, facilitating cell proliferation and invasion,and inhibiting apoptosis by regulating MZF1 expres-sion (Fig. 2f) [133]. In breast cancer too, high levelsof FTO promotes cell proliferation, colony formationand metastasis in vitro and in vivo through downreg-ulation of BNIP3 expression [134]. The compellingevidence clearly indicates a role for FTO in cancer,

Table 1 Alterations in N6-methyladenosine writers, erasers and readers in cancer. AML: Acute myeloid leukaemia; HCC:Hepatocellular Carcinoma; GSC: Glioma Stem Cells; LncRNA: long non-coding RNA (Continued)


Glioblastoma Upregulated ALKBH5 sustains oncogene expression. [136]

Gastric Cancer No change Binding to NEAT1 lncRNA, which promotes invasion and metastasis. [138]

Lung Cancer Downregulated ALKBH5 reduces oncogene expression and inhibits oncogenic miRNA. [343]

Osteosarcoma Upregulated ALKBH5 decreases the decay of the lncRNA PVT1. [344]

Ovarian Cancer Upregulated ALKBH5 enhances stability of oncogenes, self-renewal genes and survivalgenes.

[139, 142]

Pancreatic Cancer Upregulated Unknown. [140, 141]

Readers

YTHDC2 Colon Cancer Upregulated YTHDC2 upregulates expression of pro-metastatic genes. [143]

YTHDF1 Colon Cancer Upregulated Induces Wnt-β-catenin pathway and unknown. [144, 345]

Melanoma Downregulated YTHDF1 promotes the translation of tumour suppressor genes. [146]

Ovarian Cancer Upregulated YTHDF1 increases translation of oncogenes. [145]

YTHDF2 AML Upregulated YTHDF2 reduces the stability of transcripts such as TNFRSF2. [147]

HCC Upregulated YTHDF2 inhibits SOCS2 mRNA expression. [56]

HCC Downregulated YTHDF2 promotes the degradation of EGFR mRNA. [149]

Lung Cancer Upregulated YTHDF2 promotes the translation of 6PGD mRNA. [148]


yet although FTO has been described to remove m6Ain the mRNA of tumour suppressors or genes thatcould confer resistance to immunotherapy, currentlythere is insufficient evidence to confirm that the ef-fects detected in cancer are due exclusively to itsdemethylase activity.Aberrant overexpression of ALKBH5, the second m6A

demethylase to be identified, has been also associated toseveral cancer types (Fig. 2f) [135–142]. ALKBH5 hasbeen found overexpressed in glioblastoma and its ex-pression is associated with poorer prognosis, and it pro-motes GSCs proliferation and tumour progression byenhancing FOXM1 expression [136]. In breast cancer,ALKBH5 has been shown to promote tumourigenesis bydecreasing adenosine methylation in KLF4 and NANOGmRNAs, enhancing their stability in breast cancer stemcells [135, 137]. In gastric cancer, it promotes invasionand metastasis by demethylating the lncRNA NEAT1[138]. ALKBH5 expression is found increased in ovariancancer too and its expression correlates with poorer sur-vival. The upregulated expression promotes proliferation,invasion and autophagy through the mTOR and BLC2-Beclin1 pathways [139, 142]. Similar to other m6A regula-tors, ALKBH5 has been demonstrated to have a tumoursuppressive role in other tumours (Fig. 2g). For example,in pancreatic cancer cells, loss of ALKBH5 induces in-creased methylation of the lncRNA KCNK15-AS1, leadingto its downregulation and increased cell migration.ALKBH5 in pancreatic cancer also has been shown to in-hibit tumourigenesis by reducing WAF-1 levels and hin-dering Wnt signalling activation [140, 141].

Role of m6A readers in cancerm6A readers are also aberrantly expressed in cancer, andtheir defective function has been linked to oncogenic ortumour suppressive roles, however we begin to appreciateonly now the interplay of these RNA binding proteins,m6A and cancer. YTHDC2 and YTHDF1 are overex-pressed in colorectal cancer, both are associated withpoor prognosis in patients and high cell proliferation andmetastatic potential. In these tumours, YTHDC2regulates the expression of tumour promoter genes suchas HIF1A [143]. Silencing of YTHDF1 inhibits tumouri-genicity in vitro and tumour growth in vivo by inhibitingthe Wnt-β-catenin pathway and its expression induces re-sistance to chemotherapy [144]. YTHDF1 is also upregu-lated in cancer. In particular, in ovarian cancer YTHDF1increases EIF3C translation, facilitating tumourigenesisand metastasis [145]. In contrast, YTHDF1 acts as atumour suppressor in melanoma where it promotesthe translation of the tumour suppressor HINT2, thusinhibiting tumour development [146]. In the case ofYTHDF2, its overexpression in AML has been shownto reduce the half-life of various m6A-containing

transcripts which are involved in TNF signalling andwhose upregulation promotes cell apoptosis [147].Furthermore, YTHDF2 was found to be upregulatedin lung cancer, and to aberrantly promote the transla-tion of 6PGD mRNA which is critical for the promo-tion of tumour growth [148]. Nevertheless, YTHDF2is also capable to suppress cell proliferation, tumourgrowth and activation of MEK and ERK signalling viapromoting the degradation of EGFR mRNA in HCCcells [149].

Targeting m6A machinery in cancerConsidering the critical roles of the m6A regulatory pro-teins in several cancer hallmarks, they are promisingtherapeutic targets, specially the writers and erasers,since their activity can be modulated by small molecules.Although there are currently no small molecule inhibi-tors of RNA methyltransferases, several demethylaseinhibitors have been discovered or developed bybiochemical- or cell-based small-molecule compoundlibrary screening or chemical synthesis. Most commonly,those developed or discovered inhibitors target FTO. Ina seminal study, Su R et al. FTO was found to be inhib-ited by the oncometabolite R-2-hydroxyglutarate(R-2HG) [37]. In this study, R-2HG was used to directlyinhibiting FTO in AML and glioma cells, which resultedin increased methylation and decreased expression ofc-MYC and CEBPA mRNAs, blocking proliferation, cellcycle, and inducing apoptosis in AML and glioma cells[37]. Since then, other studies have attempt to developsmall-molecule inhibitors to target FTO or AKLBH5RNA demethylases, given promising results at the pre-clinical level. For example, the ethyl ester form ofMeclofenamic acid (MA) MA2, a US Food and DrugAdministration (FDA)-approved nonsteroidal anti-inflammatory drug, was found to be a FTO inhibitorwhich led to elevated levels of m6A modification inmRNAs in glioblastoma cells, suppressing tumour pro-gression and prolonging the lifespan of GSC-graftedmice [35, 150]. In addition, based on the structures ofFTO and ALKBH5 domains other groups have designed2-oxoglutarate and iron-dependent oxygenases (2OGX)inhibitors to target m6A erasers [151], such as for ex-ample the IOX3 inhibitor [152]. However the promisinginhibitors have some limitations and need to be consi-dred before their use in the clinic. All 2-oxoglutaratederivates are not selective and may also suppress the ac-tivity of other Fe (II) and 2OG dependent oxygenases.More recent studies have developed two FTO inhibitors,namely FB23 and FB23–2, which have been shown tosuppress proliferation and promote AML cell differenti-ation/apoptosis in vitro and significantly inhibit the pro-gression of human AML in xenotransplanted mice [153].


Despite the contradictory results for some types of tu-mours, m6A regulators have shown to have wide impli-cations in several cancer hallmarks. The controversialresults reflect however that the consequences of aberrantm6A deposition may dependent on the cancer or celltype context, dysregulation of other signalling pathwaysand distinct set of substrates. Thus, future studies willaccurately determine the biological function of each in-dividual m6A regulator in different types of cancer, andthey will identify each critical target transcript revealingthe exact underlying mechanism. In addition, develop-ment of selective and clinically effective inhibitors form6A regulatory enzymes may provide effective thera-peutic strategies, alone or in combination with othertherapeutic agents. For example, neoantigen-specific im-munity was shown to be regulated through YTHDF1 ina m6A-dependet manner. Mechanistically, transcriptsencoding lysosomal proteases were shown to be markedby m6A and bound by YTHDF1, leading to increasedtranslation of lysosomal cathepsins and decreased cross-presentation of dendritic cells, implicating YTHDF1 andm6A as potential therapeutic targets in anticancer im-munotherapy [116].

5-methylcytosinem5C deposition in RNAm5C is a conserved and prevalent mark in RNA in all lifedomains. m5C is found in a wide range of RNAs but it ismost abundant in eukaryotic tRNAs and rRNAs (exten-sively reviewed in [16]). Current global m5C detectionmethods rely on the chemical reactivity of cytosines tobe deaminated in the presence of sodium bisulphite, orimmunoprecipitation methods that use either antibodiesagainst m5C or RNA methyltransferases previouslycrosslinked to the RNA target (extensively reviewed in[16]). RNA bisulphite sequencing is the most commonused technique to map m5C and while it has resulted ef-fective in detecting m5C in abundant RNAs such astRNA and rRNA [24, 42]. In mRNA different studies ob-tained very different results. From studies identifyingm5C sites in 8000 RNAs [154], to other studies findingonly a few methylated mRNAs [155]. These controver-sial findings have raised the need to develop more robustmethods for truly identifying m5C depositions in mRNA[156]. More recent studies have reported only few hun-dred m5C sites in human and mouse transcriptomesusing an improved bisulphite sequencing method and anovel computational approach (Fig. 1) [156, 157]. Whatis still undetermined is whether this low prevalence hasbiological value and it needs to be further investigated.

m5C writersIn humans, cytosine-5 methylation is catalysed by theNOL1/NOP2/sun (Nsun) family and the DNA

methyltransferase member 2 (DNMT2, TRNA AsparticAcid Methyltransferase 1 or TRDMT1) [158, 159].DNMT2, NSUN2, NSUN3 and NSUN6 all methylatecytoplasmic tRNAs, yet with different specificity and atdifferent residues (Figs. 3b, 4) [42, 160–167]. For example,NSUN2 methylates the vast majority of tRNAs at the vari-able loop, and in leucine at the wobble position [42, 168,169], DNMT2 methylates three tRNAs at the anti-codonloop [161, 162, 170], and NSUN6 targets the acceptorstem of few tRNAs [164]. NSUN2 methylates also mRNA,ncRNAs and lncRNAS (Fig. 3a, c) [157, 168, 171–173].NSUN3 targets tRNAs in the mitochondria, and its depos-ition is required for the formation of 5-formylcytosine(f5m) [160, 163, 166] (Figs. 3b, 4). NOP2 (NSUN1) andNSUN5 are nucleolar and methylate very conserved resi-dues in 28S rRNA, close to the peptidyl-transferase centre[165, 174, 175] and at the interface between the large andsmall subunit respectively (Fig. 3d) [165, 174, 175]. Finally,NSUN4 targets the small subunit of mitochondrial rRNA(Fig. 3d) [176].

m5C erasersWhile writers for m5C are now well documented, the ex-istence of m5C erasers are still under debate. Some re-ports have however indicated that m5C can be oxidisedto generate 5-hydroxymethylcytosine (hm5C) by en-zymes of the ten-eleven translocator family (TET) inmRNA (Fig. 3a) [177, 178] and the formation of f5C byAlpha-Ketoglutarate-Dependent Dioxygenase AlkBHomolog 1 (ALKBH1) at the wobble position of mito-chondrial tRNAs (Fig. 3b) [160, 163, 166]. While the bio-logical relevance of f5C deposition in mitochondrialtRNAs has been well established [160, 163, 166], the bio-logical relevance of the low abundant hm5C depositionin mRNAs remains yet to be determined.

Molecular function of m5C deposition in RNARole of m5C in non-coding RNAsThe functional consequences of m5C loss in tRNAs havebeen well documented. Deposition of m5C by DNMT2and NSUN2 protects tRNAs from endonucleolytic cleav-age by angiogenin [42, 162, 167]. NSUN2-mediated m5Cdeposition in tRNAs regulates differentiation and stressresponses in tissue and in cancer stem cells [33, 42, 179–181]. Molecularly, 5-cytosine methylation of tRNA pro-tects them from processing into tRNA-derived small RNAfragments (tRFs) by angiogenin [42, 162, 167, 170, 182].The formation of tRFs is usually induced under stress andcan repress canonical translation and favour ribosome as-sembly in unconventional 5’ start sites found in 5’ UTR ofstress response transcripts [183]. In contrast, loss ofDNMT2-mediated methylation of tRNA at C38 causestRNA cleavage which leads to specific codon mistransla-tion [167]. Similarly, deletion of rRNA cytosine-5


methyltransferases in yeast, flies, worms, and mice are notlethal, however, in all cases, the level of m5C depositionplays a significant role in regulating the cellular responseto stress including drugs, DNA damage, oxidative stress,or environmental cues [23, 175, 184]. Mechanistically,rRNA modifications such as NSUN5-mediated methyla-tion fine-tune the translation capacity of the ribosome byadapting it to specifically translate mRNAs relevant instress [24]. m5C has also been detected in small ncRNAsand lncRNA [154, 168, 169, 185–187]. m5C deposition onncRNA such as vault RNA also affects its processing intomiRNA-like regulatory small RNA [168, 171].

Role of m5C in mRNAsOnly two recent studies have reported finding readersthat bind to m5C-modified mRNAs, revealing the bio-logical relevance and the role of m5C deposition inmRNAs (Fig. 3a). In one report, nuclear mature methyl-ated mRNAs at any nucleotide position interacted withthe nuclear export factor AlyREF and were more likelyto be exported to the cytoplasm [173]. More recently,another report showed that in cancer cells NSUN2 aber-rant methylation of oncogenic mRNAs at the 3′ UTR in-creased their interaction with the reader protein Y-box-binding protein 1 (YBX1), which maintained the stability

Fig. 3 Molecular mechanism of m5C deposition in RNA, biological function implication in human cancer. a-d m5C RNA deposition landscape inmRNA (a), tRNA (b), rRNA (d), and ncRNAs (c) mediated by writers (blue balloons) NSUN2–6, DNMT2 and NOP2, erasers (pink balloons) TET andALKBH1, and reader proteins (yellow balloons) YBX1 and AlyREF, and their role in mRNA metabolism. e-h Aberrant m5C deposition in RNAspromotes or suppresses tumour progression through NSUN2 upregulation in mRNA, miRNA and lncRNA (e), NSUN2 downregulation in tRNA (f),NOP upregulation in rRNA (g) or NSUN5 downregulation in rRNA (h). Red arrows indicate induction. Blue arrows with flat end represent inhibition


of its targeted mRNAs by recruiting ELAVL1 [157]. Des-pite these studies, the advances in finding the preva-lence, deposition site preference, molecular function,writers and readers of m5C on mRNA still remainelusive.

The role of m5C in cancerRole of m5C writers in cancerOver the past decade, a number of cytosine-5 methyl-transferases have been found to be associated with vari-ous human diseases including several cancer types(Table 2). NOP2 overexpression was long shown to in-crease proliferation of mouse fibroblasts (Fig. 3g) [188],and it was found to be a valuable proliferation marker[189]. NOP2 expression has been found to be upregu-lated in breast, lung, prostate cancer, and gallbladdercarcinoma, and its expression correlates with poor prog-nosis [190–192]. Mechanistically, NOP2 has been shownto bind to the T-cell factor (TCF)-binding element of thecyclin D1 promoter, recruiting TERC elements (telomer-ase RNA component) and activating cyclin D1 transcrip-tion. Whether NOP2’s methylating activity of rRNA isinvolved remains unexplored [193]. NSUN2 expression al-terations have been linked to several cancer types includ-ing breast, skin, colon, ovarian, oesophageal, bladder,gallbladder, gastric cancer, and head and neck squamouscarcinoma [157, 172, 194–200]. DNMT2 is upregu-lated in hundreds of tumour samples and severalsomatic mutations in DNMT2 have been identified indifferent tumours types [201]. NSUN4 loci is

associated to increase risk of breast, ovarian or pros-tate cancer and its high expression is associated withHCC [202, 203]. Lastly, NSUN5 expression has beenfound to correlate with poor survival in glioblastoma[24] and NSUN7 high expression is associated toshorter survival in low-grade gliomas [204].Regarding the associated mechanisms, the methylases’

molecular role seems to be tissue and contextdependent, showing different unique substrates prefer-ences for each cancer type, but with a unifying onco-genic result. For example for NSUN2, in most reports itsoverexpression is shown to regulate the fate of one sin-gle transcript of tumour suppressor genes or oncogenesand thus promoting proliferation [205]. Very recentlyNSUN2 was also shown to promote tumour progressionby methylation of NMR ncRNA in oesophageal cancer[172]. In another recent study, it was reported that aber-rant NSUN2-mediated methylation at the 3′ UTR ofoncogenic mRNAs such as heparin-binding growth fac-tor (HDGF) mRNA can increase their stability by inter-acting with YBX1 (Fig. 3e) [157]. While all these reportsfocused on the fate of mRNAs or other transcriptswhose m5C prevalence is low, and considering thattRNAs are the main NSUN2 targets and are highlymethylated at C-5, it is not yet clear whether the poten-tial role of NSUN2 in cancer might actually be mediatedby modifications of mRNA.Regarding tRNA methylation functions, NSUN2 has

been reported to be upregulated in a population ofproliferative progenitor cells in skin tumours that rely

Fig. 4 Simplified structure of a tRNA and all known Ψ and m5C events in humans. Ψ modification is shaded in blue and m5C in purple.The table on the right indicates the position and modification in tRNA, the human enzyme that has been identified to catalyse thismodification, the sub-cellular localization of tRNAs containing the modification. m5C, 5-methylcytosine; f5C, 5-formylcytosine Ψ,pseudouridine; Ψ derivates: Ψm, 2ʹ-O-methylpseudouridine and m1Ψ, 1-methylpseudouridine. Proteins: PUS, pseudouridine synthase;RPUSD, RNA pseudouridine synthase domain-containing protein; NSUN, NOL1/NOP2/SUN domain family member; DNMT2, DNAmethyltransferase 2. f5C* is formed from m5C by ALKBH1 (Alpha-Ketoglutarate-Dependent Dioxygenase AlkB Homolog 1) in mitochondrialtRNAs. Ψ derivates are formed from Ψ by methyltransferases


Table 2 Role of aberrant deposition of m5C in cancer. AML: Acute myeloid leukaemia; ALL: Acute lymphoblastic leukaemia; HCC:Hepatocellular Carcinoma


Writers

NOP2 Breast cancer Upregulated Unknown. [190]

Leukaemia Increased NSUN1 mediates chromatin structures thatmodulate 5-AZA resistance.

[216]

Lung adenocarcinoma Upregulated Unknown. [192]

Prostate cancer Upregulated Unknown. [191]

NSUN2 Bladder cancer Upregulated NSUN2 targets HDGF 3' UTR. [157]

Skin, breast and colon cancer Upregulated Unknown. [194]

Squamous cell carcinoma Upregulated Protects tRNA from cleavage and increases cell survival [33]

Gallbladder carcinoma Upregulated NSUN2 interaction with RPL6. [197]

Gastric cancer Upregulated Repressing p57(Kip2) in an m5C-dependent manner. [200]

Head and neck squamous carcinoma Upregulated Unknown. [199]

Oesophageal squamous cell carcinoma Upregulated Increased methylation and stability of NMR lncRNA. [172]

Ovarian cancer Upregulated Unknown. [195]

Several cancers Increase copy number Unknown. [198]

NSUN3 Leukaemia Undetermined NSUN3 direct binding to hnRNPK. [216]

Lung Cancer Genomic aberrations Unknown. [346]

NSUN4 Breast, ovarian, and prostate cancer Susceptibility Loci Unknown. [202]

HCC High expression Methylation processes. [203]

NSUN5 Glioblastoma Downregulation Ribosome structural changes that lead to stressadaptive translational programmes.

[24]

NSUN7 Low Grade Gliomas High expression Unknown. [204]

DNMT2 Leukaemia Undetermined DNMT2 direct binding to hnRNPK. [216]

Several cancer types Upregulated andsomatic mutations

Inhibition of m5C deposition in tRNAs. [201]

Erasers/5-hydroxymethylcytosine writers

TET1 Glioblastoma Upregulated Unrelated to RNA hydroxymethylation. [213]

TET2 Glioblastoma Downregulated Unrelated to RNA hydroxymethylation. [214]

AML Point mutations Unknown. [210]

TET3 Glioblastoma Downregulated Unknown. [215]

TET Family Hematologic malignancies Several Unrelated to RNA hydroxymethylation. [212]

TET1/TET2 HCC Downregulated Unrelated to RNA hydroxymethylation. [347]

ALKBH1 ALL Upregulated Unknown. [211]

Gastric cancer Upregulated Unknown. [348]

Readers

YBX1 Bladder cancer Upregulated YB1 translocation to the nucleus induces acquisitionof drug resistance by upregulating expression ofmultidrug resistance-1 (MDR-1) gene.

[349]

Breast cancer Upregulated YB1 interacts and inhibits ESR1-FOXA1 complex. [350]

Several cancer types Upregulated Multifunctional oncoprotein. [351]

ALYREF HCC High expression Cell cycle regulation and mitosis. [203]

Oral squamous cell carcinoma High expression Unknown. [352]

Several cancer types High expression Unknown. [353]


on the correct deposition of m5C onto tRNAs [33].Unexpectedly, deletion of NSun2 in mouse cells andmouse cancer cells led to hypomethylated tRNAswhich showed to be more vulnerable to the ribo-nuclease angiogenin, and consequently to the accu-mulation of 5′-tRNA fragments [33, 42, 182].Notably, the increase cleavage of tRNAs did not re-sulted in depletion of mature tRNAs, supporting thatcleaved tRNAs mediated the phenotypic conse-quences. In fact, there is a growing appreciation thatbiogenesis of tRNA fragments is largely inducedunder stress conditions and their aberrant expression,commonly found in cancer, may indicate importantregulatory functions in tumourigenesis (extensivelyreviewed in [183]). The understanding of their func-tion is still in its infancy, but the reported data indicatethat 5′-tRNA fragments can reprogram translation tofavour stress responses by regulating the binding of trans-lation initiation factors to the translation initiation com-plex [206–208]. Indeed, in Nsun2 deficient mouse cancercells, ribosome profiling data showed an increased transla-tion of genes associated to stress response pathways anddecreased translation of genes associated to differentiation[33]. Importantly, the data showed that Nsun2 deficiencyin cells blocked them in an undifferentiated and more pro-liferative state necessary for the self-renewal of tissue ortumour stem cells [33, 179, 181, 182]. However, prolongeddeficiency showed to increase the sensitivity to stress ofthe undifferentiated cells [33, 42]. The finding was furthersupported by showing that Nsun2 deficient skin tumourinitiating cells were more efficiently killed by using che-motherapeutic agents such as 5′ FU or cisplatin, whichcould be further rescued upon treatment with angiogenininhibitors [33]. Although the exact mechanism by which5′-tRNA fragments directly favoured translation ofspecific set of transcripts is still unknown, the studydemonstrated that the combinatory use of tRNA frag-ment biogenesis enhancers such as NSUN2 inhibitors,with convectional chemotherapeutic agents could re-sult in an efficient strategy to specifically eliminatetumour initiating cells. Thus, these and other studiesshow that tumour initiating cells and cancer cells re-quire tight control of tRNA methylation, tRNA frag-ment biogenesis and protein synthesis [209] foraccurate cell responses and to maintain the bulktumour, and suggest the use of tRNA methylation in-hibitors as potent cancer initiating cells sensitizers tocytotoxic stress (Fig. 3f).The contribution of tRNA modifications in survival

and differentiation is further supported by findings thatDnmt2 deletion in mice and flies was characterized bydefects in stress responses and differentiation [162, 167].In line with Nsun2 −/− mice phenotype, Dnmt2-depletion in mice did not globally perturb protein

synthesis rates but rather affected specific mRNAsthrough reduced translation fidelity caused by loss oftRNA methylation [167]. Yet the potential biological im-pact of elevated tRNA cleavage due to Dnmt2 loss orcytosine-5 methylation inhibition remains completelyunexplored. Additional work will be necessary to unveilthe contribution of tRNA fragment abundance to thecomplex phenotypes observed.Alterations in rRNA methylation have been also linked

to cancer. NSUN5 mRNA expression is strongly associ-ated with poor survival in glioblastoma patients. Epigen-etic loss of NSUN5 expression in gliomas leads to rRNAcytosine hypomethylation and to increased translation ofsurvival factors, rendering glioma cells sensitive to sub-strates of the stress-related enzyme NAD(P)H quinonedehydrogenase 1 (NQO1) (Fig. 3h) [24].Thus, these findings highlight the importance of the

tight control of tRNA and rRNA methylation and spe-cialised protein synthesis programmes and set the basisto search for tRNA methylase inhibitors as a potent newapproach to treat cancer.

Role of m5C erasers in cancer5-hydroxymethylation writers have been found alteredin cancer too. TET and ALKBH1 mutations or expres-sion alterations are highly associated to some malignan-cies. For example, TET2 and ALKBH1 mutations havebeen associated to lymphoblastic and myeloid leukaemia[210–212]. TET1 expression is upregulated in glioblast-omas [213], TET2 is downregulated in gliomas [214],and TET3 is epigenetically repressed in gliomas [215].While mechanistically the cause has been always associ-ated to DNA demethylation or hydroxymethylation de-fects, TET and ALKBH1 have been shown to oxidisem5C in RNA too, raising the question as to whether de-fects of RNA hydroxymethylation could be also linkedto cancer.

Targeting m5C machinery in cancerTo date no specific cytosine-5 RNA methyltransferaseinhibitor has been developed. Yet, given that severaldrugs designed to interfere with cytosine-5 methylationof DNA rely on the use of chemical analogues ofcytosine, they may well interfere with RNA methylation[50, 216]. In fact, in a study by Lyko and co-workers,complete inhibition of Dmnt2-mediated 5-cytosinetRNA methylation with azacytidine in cancer cells re-duced their proliferative capacity, supporting the notionthat reduced cytosine-5 methylation of tRNAs may bean efficient cancer therapeutic strategy [50]. Despite thepromising results and potential clinical implications, thefact that those analogues can both inhibit RNA andDNA methyltransferases raise awareness of the use ofthis unselective drugs, which may affect the methylation


of multiple targets (DNA, tRNA, rRNA, mRNA, ncRNA)and may have devastating consequences.In line with the notion that inhibition of tRNA meth-

ylases may contribute to chemotherapy resistance,silencing of other tRNA methyltransferases such as the7-guanosine methylase METTL1 which also methylatestRNAs at the variable loop of several tRNAs has shownto increase sensitivity of cancer cells to 5’FU [217].

PseudouridinePseudouridine deposition in RNAPseudouridine (Ψ), the C5-glycoside isomer of uridine,was the first posttranscriptional modification discoveredand is one of the most abundant modifications of RNA[218, 219]. Despite its discovery over seventy years ago,we are only now beginning to uncover its biologicalfunction [25–27, 220–223].Pseudouridine was initially detected on yeast tRNAs

and rRNA [224, 225], and now we know that differenttypes of RNAs including tRNA, rRNA, small nuclearRNAs (snRNAs), small Cajal Body-specific RNAs(scaRNAs), miRNAs, lncRNAs are also Ψ-modified[226]. Most importantly, with the current technologicaladvents which rely on chemical treatment of RNA usingsoluble carbodiimide(N-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-ρ-toluenesulfonate (or CMCT) for thegeneration of reverse transcription-stops, several groupshave successfully performed genome-wide mapping experi-ments validating established targets and revealing novel sub-strates like mRNAs (Fig. 1) [25, 26, 221]. Since then, severalseminal studies have followed discovering novel substratesand molecular roles of Ψ [25–27, 221, 222, 227–230].

Pseudouridine synthetasesPseudouridylation can be achieved through two distinctmechanisms, namely RNA-independent and RNA-dependent pseudouridylation. The RNA-dependentmechanism relies on RNA–protein complexes known asbox H/ACA small ribonucleoproteins (snoRNPs), whichconsist of a box H/ACA snoRNA and four core proteins;dyskerin (also known as NAP57 or DKC1), non-histoneprotein 2 (Nhp2), nucleolar protein 10 (Nop10) andglycine-arginine-rich protein 1 (Gar1). In H/ACAncRNA is responsible for substrate recognition throughcomplementary base-pairing interactions with the RNAsubstrate, and the catalytic activity is provided by DKC1(Fig. 5a) (reviewed in [227]). The RNA-independentpseudouridylation is catalysed by a single enzyme,pseudouridine synthases (PUS), which carry both sub-strate recognition and catalysis without an RNA tem-plate strand (Fig. 5b). The rules governing RNAsubstrate recognition by RNA-independent Ψ synthaseshave only been elucidated in a few cases (for a review,see [231]). Some of the characterized Ψ synthases have a

rather strict substrate specificity and are only able tomodify one position in only one type of cellular RNA,such as, for instance, tRNAs. The strict substrate specifi-city depends on the universally conserved G53UUCNANNC60 sequence in most tRNAs, and on the three-dimensional structure of the TΨC loop [232]. Similar re-sults were found by analysing the co-crystal structures ofthe prokaryotic enzyme TruB with isolated tRNA stem-loops [233]. The crystals illustrated how the enzyme coredomain makes extensive interactions with the RNA, andhow TruB requires the consensus sequence around theTΨC loop [233]. The mechanism for enzymes with abroader susbtrate spectrum is different. For example,PUS1, which methylates three positions in tRNAs, relieson two positively charged RNA-binding clefts along thesurface of the protein which uniquely interacts with thetRNA [234]. In the case of PUS7, the sequence and two-dimensional structure are required for substrate recogni-tion [235]. How other Ψ synthases with a broader sub-strate repertoire can recognize all the different substrateswith a high degree of specificity is still under study.In eukaryotes, there are over fourteen different PUS en-

zymes, ten of these enzymes belong to the PUS family, in-cluding PUS1-10 [227, 236]. Each of them has specificsubstrates, but also share some of the substrates (Fig. 5C).For example, PUS1 modifies tRNAs at positions 38, 39and/or 40 [234]. PUS4 [237] and PUS10 modify tootRNAs, but at position 55 [238]. Regarding erasers, an im-portant difference between pseudouridylation and basemethylations is that pseudouridylation is an irreversiblemodification in mammals, and instead mammals excretethe intact nucleoside [239]. No readers have been de-scribed to date.

Molecular function of Ψ deposition in RNAPseudouridylation plays different physiological roles de-pending on the RNA that is modified, but most com-monly experimental data confirmes an important role indifferent aspects of gene expression regulation. Thepresence of Ψ is capable of increasing the rigidity of thephosphodiester backbone of the RNA and affecting itsthermodynamic stability and spatial conformation, makingshort RNAs more stable [240, 241]. In snRNAs, in vitropseudouridylation generates RNA conformational changesthat influences the snRNA-mRNA interactions [242, 243].In vivo studies have shown that these conformationalchanges are important for the snRNA activity. For ex-ample, the yeast U2 snRNA is pseudouridylated duringstress at positions U56 and U93 by the RNA-independentenzyme PUS7 and a box H/ACA RNP complex (snR81),having functional implications in the efficiency of pre-mRNA splicing [229]. Ψ’s role in small nuclear RNAs wasthoroughly reviewed recently in [220].


Pseudouridylation also generates structural stability todifferent types of RNA, such as rRNAs, which is neces-sary for their function [244, 245]. In rRNAs, Ψs arefound at the decoding site, mRNA channel, peptidyltransferase centre, tRNA binding site and ribosomal sub-unit interface, thus showing an important role for thecorrect assembly and function of the ribosome and forprotein synthesis [246]. For example in yeast, alterationsor substitutions of amino acids in the Ψ synthase do-main of Cbf5p abolish pseudouridylation of rRNA,resulting in growth defects and reduced levels of cyto-plasmic 40S and 60S subunits of rRNA [244]. In mouseembryonic fibroblast cells, expression of dyskerin mu-tants leads to altered rRNA processing, unstable second-ary structure of rRNA and cell growth defects [245].Another similar example occurs upon the loss or disrup-tion of H/ACA snoRNAs that guide rRNA modification.Pseudouridylation at position U2919 in yeast rRNA is

guided by five H/ACA snoRNAs, whose loss leads to re-duced rRNA pseudouridylation at U2919, and impaired18S rRNA biogenesis and growth defects in yeast [247].In mRNAs, the incorporation of Ψ can mediate

nonsense-to-sense codon conversion facilitating the basepairing in the ribosome decoding centre, and thus result-ing in protein diversity [248]. Other in vitro studies havealso reported that translation of mRNAs containing pseu-douridines is slowed down and mRNA decoding affectedcompared to non-modified mRNAs [249]. In addition, Ψ-containing mRNAs have shown higher stability in cellsundergoing stress, suggesting that increased pseudouridy-lation increases their stability [25–27]. In fact, in vitro-transcribed mRNAs containing pseudouridines that wereintroduced in mammalian cells or into mouse tissues dis-played enhanced stability relative to uridine-containing invitro-transcribed mRNA [250]. Notably, the majority ofpseudouridines in mRNA are regulated in response to

Fig. 5 Ψ deposition mechanisms and pathological implications in cancer. a Schematic illustration of RNA-dependent pseudouridylationmechanisms. Substrate recognition is achieved by sequence and structure homology of the substrate with the structural stems and loops formedby box H/ACA small nucleolar RNAs (snoRNA). Dyskerin (DKC1) is the catalytic unit and non-histone protein 2 (Nhp2), nucleolar protein 10(Nop10) and glycine-arginine-rich protein 1 (Gar1) are regulatory units. The RNA substrate is represented in green. b Illustration of RNA-independent pseudouridylation mechanisms. Substrate recognition and catalysis are performed by one single pseudouridine synthases (PUS)(blue). c Representative scheme illustrating the target specificity for each pseudouridine synthases. The fate of each modified RNA is alsoillustrated with orange arrows. Pseudouridylated RNAs may be also recognised by still unknown readers (?). d Altered expression of pseudouridinesynthases can lead to cancer. For example, reduced expression of DKC1 induces a reduction of pseudouridylation in TERC and rRNA, leading todysfunctional TERC and rRNA and increasing tumourigenesis. e In glioma, An increased expression of DKC1 can lead to an increased Ψ depositionon rRNA, snRNA and TERC, and thus promoting cancer cell growth and migration. f PUS7 decreased expression leads to hypomodified tRNA-derived fragments, leading to increased self-renewal and survival in bone marrow mononuclear cells, promoting tumourigenesis. Red arrowsindicate an increase and blue arrows a decrease of processes or enzymes


environmental signals, but its functional role is still poorlyunderstood [25–27, 220–223].In tRNAs, pseudouridylation is found at the anticodon

stem and loop, at the D stem and in a conserved at theΨ loop, position 55, all of which stabilize its tertiarystructure and are important for codon–anticodon base-pairing [227] (Fig. 4). Novel insights into the role oftRNA pseudouridylation were recently obtained usinghuman embryonic stem cells (hESCs) and haematopoi-etic stem and progenitor cells (HSPCs) [230]. This workunveiled that similar to m5C deposition at the anti-codon and variable loops, the deposition of Ψ at theeighth position of tRNAs by PUS7 regulated the biopro-duction of a specific class of 18-nt 5′ tRNA fragmentscontaining a 5′ terminal oligoguanine motif (mTOGs).In addition, the study showed that the presence of Ψ atposition 8 of this novel class of small ncRNAs was re-quired for efficient biding to polyadenylate-binding pro-tein 1 (PABPC1), an integral component of the 5′ captranslation initiation complex, resulting in displacementof PABPC1 from capped mRNAS and reprogramingtranslation [230]. Thus, the study showed that RNAmodifications, by regulating the biogenesis of a novelclass of ncRNAs, they can specialized the translationmachinery.The role of Ψ on other RNAs remains still poorly

understood. For instance, TERC is also pseudouridylated[26], where two specific sites of pseudouridylation havebeen detected, although there is currently no evidencethat this modification may be involved in telomerase ac-tivity [251]. In miRNA, depletion of PUS10 results in amarked reduction of the expression levels of maturemiRNAs and concomitant accumulation of unprocessedpri-miRNAs, but unexpectedly, this process results inde-pendent of the catalytic activity of PUS10 [228].In sum, Ψ deposition may confer different molecular

properties to the modified RNAs, which changes theirfate or activity. While most recent studies are focusedon Ψ modifications in mRNA, yet its role on mRNA isstill unclear. Thus, further studies will be required tofully understand the molecular role of Ψ on mRNAs. Ψis highly abundant on other RNA types such as snRNA,rRNA or tRNA, and whose role has been studied formany years. Yet, recent findings are still revealing noveland unexpected functions, such as the role of Ψ ontRNA fragments, and thus indicating that other func-tions are still to be discovered.

Role of pseudouridylated RNAs in cancerRole of DKC1 in cancerOne of the most studied disease linked to defects inpseudouridylation is the X-linked Dyskeratosis Conge-nita (X-DC), associated to DKC1 inactivating mutations[252]. Dyskerin modifies mainly rRNAs and snRNAs and

it also participates in the active telomerase complex[219]. X-DC is characterized by defects in reticulate skinpigmentation, nail dystrophy, and mucosal leukoplakia,but bone marrow failure is the principal cause of earlymortality in X-DC patients [253]. Patients with X-DCare also characterized by having higher risk for cancerdevelopment, and expression alterations are associatedto cancer too [254]. DKC1 alterations have been foundassociated to skin cancer [255], breast [256, 257], colon[254, 258, 259], lung [254, 260], prostate [261], head andneck [262, 263], glioma [264], HCCs [265], and speciallybone marrow failure syndromes and hematologic malig-nancies including chronic lymphocytic leukaemia [266,267] or multiple myeloma [268–270] (Table 3). Fromthe molecular point of view, DKC1 is a nucleolar proteinthat participates in the stabilization of the telomeraseRNA component, necessary for telomerase activity [271,272], and pseudouridylation of diverse rRNA residues atimportant ribosome domains for tRNA and mRNAbinding, all of which are important functions in highlyproliferating cells [273]. Thus, lack of dyskerin activitycauses a reduced replicative potential and prematureageing by an impairment of telomerase activity [251]and impediment of ribosome translation of specificmRNAs [274, 275], primarily affecting tissues with rapidcell turnover (Fig. 5d). Mechanistically, it has beenshown that low levels of pseudouridylated rRNA down-regulates the internal ribosome entry (IRES)-dependenttranslation of tumour suppressors such as p53 [276],p27 and inhibitors of apoptosis (Bcl-xL, XIAP) [274,277]. Increased expression of vascular endothelialgrowth factor has been associated to loss of DKC1 [278],resulting their depletion in high incidence of cancer de-velopment. Other recent studies investigating the role ofΨ in rRNA have also supported the emergent hypothesisof the existence of specialized ribosomes in cancer. Inliver cancer cells, aberrant expression of snoRNA24,which mediates the pseudouridylation at U609 and U863positions in 18S rRNA, leads to changes on tRNA selec-tion efficiency, ribosome elongation rate and translationefficiency influencing cancer cell survival [279]. Otherexample of pseudouridine modifications in rRNA is thereduction of m1acp3Ψ modification of rRNA found incolorectal carcinoma, which affects the direct interactionof tRNA with ribosomal P site, altering and deregulatingtranslation in cancer cells [280]Although there is more evidence that Dyskerin acts as

a tumour suppressor [274, 277, 278, 281], in contrast,other studies have indicated an oncogenic role. For ex-ample, in breast cancer, decreased levels of DKC1 ex-pression, rRNA pseudouridylation and telomere lengthcorrelate with better prognosis [256]. In glioma, in-creased expression of DKC1 and pseudouridylation pro-mote glioma cell growth and migration by inducing the


upregulated expression of gliomagenesis regulators, al-though the direct role of increased pseudouridylation ofRNAs and gliomagenesis remains unexplored (Fig. 5e)[264].The data so far show that alterations in DKC1 expres-

sion or activity are significantly associated to cancer,however the exact mechanism may be cell, tissue orDKC1 substrate dependent. Further studies will be re-quired to fully explore whether DKC1 has a pro-oncogenic or tumour suppressive role.

Role in cancer of RNA-independent pseudouridinesynthetasesDespite the significant association of DKC1 activity orexpression alterations to cancer, little is known on therole of other pseudouridylases and only few studies haveassociated alterations on their expression or activity tocancer (Table 3). For example, PUS1 mediates the inter-action of steroid receptor RNA activator 1 (SRA1) with

retinoic acid receptor-γ (RARγ) in melanoma cells, andwith oestrogen receptor in breast cancer cell lines [282].PUS10 is a mediator of TNF-related apoptosis inducingligand (TRAIL)-induced apoptosis in prostate cancercells [283], and genomic alterations of PUS10 locus aresignificantly associated with lung cancer risk [284], al-though it is not clear whether this effects are dependenton pseudouridylase activity. Loss of PUS7 occurs fre-quently in myelodysplastic syndromes, haematologicalclonal disorders characterised by haematopoietic stemcells dysfunction and high risk of transformation toAML [285]. Mechanistically, Guzzi et al. demonstratedthat PUS7 depletion in hESC and HSPCs reduced PUS7-mediated pseudouridylation in a special class of tRNA-derived RNA fragments, inducing significantly higherprotein synthesis rates leading to dramatic growth anddifferentiation defects (Fig. 5f) [230]. Thus this worksupports the emerging view that ESCs and cancer cellsare highly sensitive to perturbations of protein synthesis,

Table 3 Role of pseudouridylases in cancer. AML: Acute myeloid leukaemia; CLL: Chronic lymphocytic leukaemia; HCC:Hepatocellular Carcinoma

Factor/Enzyme Cancer type Regulation Mechanism Ref

Writers

DKC1 Breast cancer Downregulated DKC1 downregulation leads to an impairment of hTRstabilization, telomerase activity and proper rRNApseudouridylation.

[256, 257]

CLL Downregulated Lower telomerase activity and lower expression ofsheltering components, which facilitates telomericdamages.

[266]

Colorectal and lung cancer Sporadic mutations Unknown. [254]

Colorectal cancer Upregulated DKC1 increases the expression of TERC and rRNApseudouridylation, promoting proliferation.

[258, 259]

Glioblastoma Upregulated DKC1 upregulates the expression of N-cadherin,MMP-2, HIF1A, CDK2 and cyclin E.

[264]

Head and neck cancer Upregulated Unknown. [262, 263]

HCC Upregulated Unknown. [265]

Lung cancer Upregulated High levels of TERC, leading an increasedaggressiveness and poor prognosis.

[260]

Multiple myeloma Genomic mutation Telomere length and expression levels of smallnucleolar and small Cajal body-specific RNAs.

[268–270]

Prostate cancer Upregulated Increased abundance of several H/ACA snoRNAs. [261]

Skin cancer Sporadic mutations Unknown. [255]

Pituitary cancer Sporadic mutations Defected translation of specific mRNAs harbouringinternal ribosomal entry site (IRES) elements,including the tumour suppressor p27.

[277]

PUS1 Melanoma and breast cancer No change Interaction of steroid receptor RNA activator 1(SRA1) with retinoic acid receptor-γ (RARγ) in melanomacells and with oestrogen receptor (ER) in breast cancercell lines.

[282]

PUS7 Myelodysplastic syndromes/ AML Loss Reduced bioproduction of tRNA-derived fragmentsleading to significantly higher protein synthesis.

[230, 285]

PUS10 Prostate cancer No change PUS10 is a coactivator of TNF-related apoptosisinducing ligand (TRAIL)-induced apoptosis.

[283]

Lung cancer Genomic alterations Unknown. [284]


and highlights an important role for tRNA fragment inreprograming translation [286]. Additional work usingin vivo models and patient-derived cancer cells will benecessary to explore the clinical implications of targetingPUS7 loss or aberrant tRNA fragment bioproduction incancer.

Targeting pseudouridine synthetases in cancerFrom the clinical point of view, PUSs or Ψ may serve aspotential anti-cancer targets and biomarkers. For in-stance, high amounts of Ψ have been detected in urineof colon, prostate or ovarian cancer patients, plasma ofovarian patients or in salivary metabolites of oral squa-mous cell carcinoma patients, suggesting to be a poten-tial biomarker in liquid non-invasive biopsies for earlycancer diagnoses [261, 287–290]. Yet, while mutationsand expression alterations in DKC1 have been signifi-cantly reported in cancer, little has been reported on thestatus of other Ψ synthetases. With the advent of cancergenome- and transcriptome-wide studies, future compu-tational analyses will reveal the mutational and expres-sion status of all known human Ψ synthetases.Regarding the design of drugs or screening for small

molecules that inhibit PUSs activity, while several studieshave attempted to generate or find compounds to di-minish DKC1 activity as potential anti-cancer treat-ments, very little progress has followed [291–293].Clinical trials were performed in ovarian carcinoma[294], sarcoma [295], colorectal carcinoma [296], acutemyelogenous leukaemia [297], breast cancer [298], lungcancer [299], melanoma [300] and other cancers [301] toexamine the effectivity as anti-cancer therapy of pyrazo-furin, a small molecule inhibitor of the orotodine-5′-monophosphate-decarboxylase (ODCase) which alsoinhibits DKC1. In all cases pyrazofurin failed to demon-strate efficient anti-cancer activity. However, whetherpyrazofurin could be an effective treatment for patientswith overexpressed DKC1 was not concluded from thosestudies, since DKC1 expression levels were not takeninto account.Another potential small molecule inhibitor that is

already used as effective anti-cancer agent in the clinic is5’FU. Treatment with 5’FU is known to improve survivalin various cancers [302]. While the mechanism of actionfor active metabolites of 5’FU has been always attributedto disruption of both DNA and RNA syntheses andDNA damage induction [302], 5’FU was shown to inhibitpseudouridine synthases, due to the substitution of ura-cil by the analogue 5’FU in RNAs [303, 304]. In thosestudies, Samuelsson et al. demonstrated that the use offluorinated tRNAs generated specific and stable non-co-valent complexes with yeast pseudouridine synthases,thus acting as potent and irreversible inhibitor [304].Nonetheless, 5’FU is not an universal inhibitor for all

pseudouridine synthases, in fact TruB E. Coli, and mostlikely its eukaryotic homologues, cannot form covalentadducts with fluorinated RNAs [303]. Thus, it would beinteresting to test the specificity and efficacy of 5’FU ininhibiting mammalian pseudouridine synthases. Inaddition, it would be important to determine whetherpart of the associated cytotoxicity of 5’FU is due to anoverall decrease of RNA pseudouridylation or to the lossof a particular modified RNA (e.g. rRNA).More recent studies have used in silico approaches

which can predict possible new inhibitors. One studypredicted the use of a small molecule inhibitor based onthe disruption of the interaction of DKC1 with TERC[293]. The virtual docking-based screen found ten mole-cules with high affinity values, of which three resulted aspotent telomerase inhibitors in a breast cancer cell line.In another study, Floresta et al. hypothesized that nu-cleoside analogues such as the isoxazolidinyl derivative5′-monophosphate could act as an inhibitor of pseudo-uridine 5′-monophosphate glycosidases competing withthe natural substrate and hampering the glycosidic C–Cbond cleavage [292]. They indeed found that the isoxa-zolidinyl derivative accommodated within the active siteof the enzyme with higher ligand efficiency than the nat-ural substrate, leading to the enzyme inhibition in vitro.While we still ignore the tumour growth inhibitory po-tential and the therapeutic benefits of using those firstinhibitors, these studies set the basis to continue withthe search for Ψ synthetase inhibitors for cancertreatment.

ConclusionRNA modifications have emerged as critical posttranscrip-tional regulators of gene expression programmes. We startto appreciate the functional networks that the epitran-scriptome interacts with, ranging from metabolisms [31]to epigenetics and chromatin remodelling [24, 216] or theimmune system [116]. Despite the progress, most studieshave focused on the molecular and physiological functionsof only one mark, 6-methyladenosine on mRNA, howeverthe epitranscriptome embraces over 170 RNA chemicalmodifications that decorate coding and ncRNAs, severalother posttranscriptional RNA processing events, andRNA binding proteins that may be as well modified as his-tones in DNA [1]. Thus, association studies of hundredsof other RNA marks on coding but also ncRNAs such astRNA or rRNA remain to be explored.To achieve this, we first need to develop system-

wide methods and tools for rapid and quantitative de-tection of RNA modifications. Most of the stablishedmethods rely on next-generation sequencing and, assuch, they are typically blind to nucleotide modifica-tions. Consequently, indirect methods are requiredthat are based on immunoprecipitation techniques


using specific antibodies, or enzymatic methods andchemical labelling and unique base-modification prop-erties of RNA pairing [305]. These methodologieshave allowed us to catalogue and identify endlessmodifications with high precision and at nucleotideresolution, yet these strategies have some limitations,reproducibility rate is low due to technical limitationsand poor computational methods. For example, anti-bodies used to recognize modifications such as m6Astill exhibit non-specific binding and can bind to N6,2′-O-dimethyladenosine (m6Am) too [59]. In fact, allantibody-related approaches suffer from poorly char-acterized, and thus unpredictable specificity of theantibody used for enrichment [306]. To overcomethese limitations, novel detection methods have beenintroduced such as DART-seq (deamination adjacentto RNA modification targets), an antibody-freemethod for detecting m6A sites, where the cytidinedeaminase APOBEC1 is fused to the m6A-bindingYTH domain [307]. Yet, this methodology is also lim-ited since it allows to simultaneously map only RNAsbound to YTH domain containing readers. For m5Cdetection, bisulphite-RNA sequencing is the goldstandard, yet the reproducibility is low, especially inlow abundant and unstable RNA such as mRNAs[16]. The large initial amount of RNA required tocompensate for the high losses caused by thetreatment, the resistance to C-U conversion fromneighbouring modifications or double-strandedsequences, the inability to differentiate from othermodifications protecting the C from the conversationsuch as 5-hydroxymethylation or 3-methylcytosine,and poor computational analysis are the most com-mon found difficulties [16]. Yet, careful assessment ofC-U conversion together with development of statisti-cally robust bioinformatic tools that highly refined fordata analysis are still generating contradictory results[155, 156]. Regarding the detection of pseudouridine,several labs have developed methodologies that relyonly the chemical treatment of RNAs with CMCT,resulting in little overlap of pseudouridine sites onmRNA from the different studies [25, 26, 221]. Thus,despite to the variety of the established techniques,the reality is that there is currently no generic andprecise method for mapping and quantifying modifi-cations in RNA. In addition, current methods arecomplex and lack of single molecule resolution. Inthis regard, the emerging third-generation sequencingtechnologies, such as the platforms provided by Ox-ford Nanopore Technologies (ONT) and Pacific Bio-sciences (PacBio) have been proposed as a new meansto directly detect RNA modifications [308]. RNAmodifications can be detected by kinetic changes ofreverse transcriptases when encountering a modified

nucleotide (PacBio) [309]. Or by current changes asthe native RNA molecule is pulled through a mem-brane pore (ONT) [310]. Although the detection ofmodifications using ONT direct RNA sequencing isalready a reality [311], yet current efforts have notyielded an efficient and accurate RNA modificationdetection algorithm, largely due to the challenges inthe alignment and re-squiggling of RNA current in-tensities. But emerging alternative base-calling strat-egies such as EpiNano algorithms which identifiesm6A from RNA reads with an overall accuracy of~90%, open new avenues to explore additional RNAmodifications in the future [312].The dynamic expression patterns of writer, reader and

eraser proteins complicate the identification of the pre-cise functional consequences of aberrant deposition ofmodifications on RNA metabolism. Thus uncovering thecomplete repertoire of cellular RNA substrates and thewriters, readers, and erasers will unveil how the intricatenetwork of epitranscriptomic events can converge intosimilar cellular processes and showing how their unbal-anced deposition may lead to pathologies. For example,HIF1A mRNA is stabilized by m6A deposition, while inmelanoma cells high levels of HIF1α protein are main-tained by modifications at the U34 wobble position oftRNAs [51, 143]. Furthermore, it will be essential tounderstand the factors or signals that determine the spe-cificity of the RNA modification writers, readers, anderasers and how these proteins are regulated in differentcell types. For instance, METTL16 is sensitive to SAMlevels and can regulate its synthesis by modifying theSAM synthase gene MAT2A as a feedback loop mechan-ism [313]. In addition, we need to develop innovativetechnologies for precise manipulation of the epitran-scriptome and functional assays that enables to under-stand their dynamic mechanisms of action of eachmodified RNA, since depletion of individual RNA modi-fier may not be sufficient to comprehensively understandtheir roles. For example, while the main target forNSUN2 are tRNAs, it still remains unclear whether thephenotypic changes seeing upon Nsun2 deletion arecaused by decrease methylation of tRNAs, or other RNAsubstrates may as well contribute to the observed pheno-type [42, 168, 179, 181, 182].We start to appreciate the wide range of functional

consequences of the aberrant deposition of RNA modifi-cations in human diseases including cancer. For ex-ample, aberrant deposition of tRNA modifications,including Ψ and m5C, leads to perturbed accumulationof tRNA fragments, a novel class of functional ncRNAswhose role is associated with aberrant protein synthesisrates and reprograming of the translational machinery intissue and cancer stem cell populations [33, 230]. Thesefindings support the current view that balanced protein


synthesis and tight control of mRNA translation is cen-tral to cellular processes involved in tumorigenesis [314]and highlights a key role for tRNAs and tRNAs frag-ments in tumourigenic processes [315]. Yet, our under-standing on how specific species of tRNA fragmentsgovern these processes and the clinical implications oftheir aberrant expression are still very limited. Futurestudies will be necessary to decipher the molecular basesof the translation reprograming driven by tRNA frag-ments. Additional work will be necessary to differentiatethe contribution of global changes in tRNA pools versusspecific population tRNA-derived fragments in thetumour promoting effect of aberrant protein synthesis[315]. More importantly, given their association to can-cer progression, their therapeutic potential must be ex-plored exploiting the current advent in miRNA-basedtherapeutics [316].Recent advancements in the rapidly evolving field of

epitranscriptomics have linked the reprogramming ofcomponents of the epitranscriptomic machinery includ-ing writers, erasers or readers of the m6A, m5C or Ψ tocancer. The extensive number of RNA modificationsthat constitute the epitranscriptome and the reversiblenature of epitranscriptomic aberrations hold promise tothe emergence of a promising field of epitranscriptomictherapy, which is already making progress with the re-cent development of effective inhibitors against m6Amodifying proteins. In the last years, several seminalstudies have shed light onto the diagnostic and thera-peutic potential of targeting the cancer epitranscriptomiccode [3, 33, 317], yet we are far from understandingwhether spatio-temporal modifications of the epitran-scriptome can drive tumour initiation [318]. In addition,their use as therapeutic agents remains a great challengedue to the lack of consistent and consolidated evidenceon the oncogenic or tumour suppressive nature of forexample the aberrant deposition of m6A. The inconsist-ent evidence may reflect the precise functional outcomeof the RNA modification on each modified RNA type,their crosstalk with other active signalling processes andthe dynamic nature of the epitranscriptome. Identifyingaccurate epitranscriptome biomarkers and defining theoncogenic or tumour suppressive effect of a given aber-rant modification within a specific cellular context, celltype, cellular proliferative capacity or tumour micro-environment will guide finding the exact molecular tar-gets to develop selective and effective therapies for agiven tumour type.Though aberrant expression of several methylases and

Ψ synthetases has now been described in cancer, it re-mains unclear whether they could be efficient targets forcancer therapy. Thus, the precise contribution of meth-ylases and Ψ synthetases to tumour initiation, growth,metastasis and resistance need to be further investigated.

It remains unclear the RNA target specificity of each en-zyme and how specific modified targets can contributeto the malignant phenotype. In addition, little is knownon the dynamics of the deposition of these modifica-tions, their erasers and how the binding to their readersinfluence the RNA metabolism and tumour cell fate.Yet, the availability of the 3D structure of most of theseenzymes and the fact that potent and selective inhibitorshave been found [52], it is reasonable to expect that in-hibition of these enzymes is achievable. These structurescan provide the basis for structure-guided drug designwhich in combination with computational tools can bepowerful resources for the development of RNA modify-ing enzymes inhibitors. Few small molecule inhibitorshave been described that can target specifically m6Aerasers, yet none of them have reached clinical stages[52]. For m5C methylases, azacytidine and decitabine(5-aza2′-deoxycytidine), which are cytidine analoguesand can inhibit any cytosine-5 methylase, have been ap-proved for clinical use in haematological malignancies[319]. However, their use should be taken carefully dueto the lack of specificity of these inhibitors that can in-hibit both RNA or DNA cytosine methylases. The valid-ation of these enzymes and their modifications as goodpharmacological targets will require the discovery of po-tent, selective, cell-permeable inhibitors to determinethe therapeutic benefit and potential risks associatedwith inhibiting these enzymes.Treatment failure in certain settings has been attrib-

uted to the presence of sub-populations of cancerstem cells or persister cells which are intrinsically re-sistant to many therapeutic approaches [320]. Giventhat m6A, m5C or Ψ regulate cell survival to stress inmany settings and stem cell functions, targeting themrepresent a very promising opportunity to specificallytarget these cells populations and reduce chemoresis-tance and recurrence. Whether other epitranscrip-tomics changes can lead to drug resistance is not yetunderstood. This clearly opens the opportunity to ex-plore novel avenues to develop diagnostic tools basedon epitranscriptomic signatures that allow for betterpatient stratification. In addition, combinatorial thera-peutic strategies with the potential to re-establish thenormal epitranscriptomic landscape or inhibit survivalsignalling pathways are promising strategies to specif-ically eliminate cancer stem cells. Combinatorial ther-apies that target independent pathways may be abetter option as the possibilities for the developmentof tumour drug resistance may be more limited. Un-derstanding the machineries and factors that intro-duce, remove, or read RNA modifications will allowthe development of novel drugs with pharmaceuticalvalue, not only for cancer but other complex humanpathologies that have been linked to aberrant


deposition of RNA modifications such as diabetes,neurological, immune and mitochondrial-linked disor-ders [47].

Abbreviations6PGD: 6-Phosphogluconate dehydrogenase; ADAM19: ADAMmetallopeptidase domain 19; AFF4: AF4/FMR2 family member 4; AKT: AKTserine/threonine kinase; ASB2: Ankyrin repeat and SOCS box containing 2;AXL: AXL receptor tyrosine kinase; BCL-2: B cell leukaemia 2; BNIP3: BCL2interacting protein 3; MET: MET proto-oncogene, receptor tyrosine kinase;CDK2: Cyclin dependent kinase 2; CEBPA: CCAAT enhancer binding proteinalpha; CMCT: Carbodiimide(N-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-ρ-toluenesulfonate; CXCR4: C-X-C motif chemokinereceptor 4; E2F1: E2F transcription factor 1; EGFR: Epidermal growth factorreceptor; EIF3C: Eukaryotic translation initiation factor 3 subunit C; ELAVL1: ELAV like RNA binding protein 1; ERCC1: ERCC excision repair 1;ERK: Mitogen-activated protein kinase 1; ESR1: Estrogen receptor 1;FOXM1: Forkhead box M1; GLI1: GLI family zinc finger 1; HBXIP: Hepatitis Bvirus X-interacting protein; HIF1A: Hypoxia inducible factor 1 subunit alpha;HINT2: Histidine triad nucleotide binding protein 2; ITGA6: Integrin subunitalpha 6; ITGB1: Integrin subunit beta 1; JUNB: JunB proto-oncogene, AP-1transcription factor subunit; KCNK15-AS1: KCNK15 and WISP2 antisense RNA1; KLF4: Kruppel like factor 4; LATS-2: Arge tumor suppressor kinase 2;LEF1: Lymphoid enhancer binding factor 1; Let-7 g: MicroRNA let-7 g; MALAT1: Metastasis-associated lung adenocarcinoma transcript 1; MEK: Mitogen-activated protein kinase kinase 7; MMP2: Matrix metallopeptidase 2;mTOR: Mechanistic target of rapamycin kinase; MYB: Myeloblastosisoncogene; MYC: Myelocytomatosis oncogene; MZF1: Myeloid zinc finger 1;NANOG: Nanog homeobox; NEAT1: Nuclear paraspeckle assemblytranscript 1; NF-κB: Nuclear factor kappa B subunit 1; NMR: Long intergenicnon-protein coding RNA 1672; NOP2: NOP2 nucleolar protein; NSUN: NOP2/Sun RNA methyltransferase; P2RX6: Purinergic receptor P2X 6; P53: Tumorprotein p53; PDCD1: Programmed cell death 1; PI3K: Phosphatidylinositol 3-kinase,; Pri-miRNA: Primary microRNA; Pre-miRNA: Precursor microRNA;PRMT1: Protein arginine methyltransferase 1; PTEN: Phosphatase and tensinhomolog; RARA: Retinoic acid receptor alpha; SETD7: SET domain containing7, histone lysine methyltransferase; SHH: Sonic hedgehog signaling molecule;SOCS2: Suppressor of cytokine signaling 2; SOX10: SRY-box transcriptionfactor 10; SOX2: Sex determining region Y box 2; SPRED2: Sprouty relatedEVH1 domain containing 2; TAZ: Tafazzin; TERC: Telomerase RNA component;TET: TET methylcytosine dioxygenase; TNF: Tumor necrosis factor; TNFRSF2: TNF receptor superfamily member; VASH: Vasohibin 1; WAF-1: Cyclindependent kinase inhibitor 1A; XIAP: X-linked inhibitor of apoptosis; XIST: Xinactive specific transcript; YAP: Yes1 associated transcriptional regulator

AcknowledgementsWe apologise for not being able to cite all the relevant publications due tospace limitations. We acknowledge funding from the Spanish Ministry ofEconomy and Innovation (MINECO), Ministry of Science and Innovation andAgencia Estatal de Investigación (AEI) and co-financed by the EuropeanDevelopment Regional Fund (FEDER) under grant no. SAF2016-78667-R (AEI/FEDER, UE) and PID2019-111692RB-I00. In addition we acknowledge fundingfrom The Scientific Foundation AECC under grant no. LABAE19040BLAN andthe University of Salamanca and Fundación Memoria de Samuel SolorzanoBarruso (FS/21-2018). BML was supported by Junta de Castilla y León-FondoSocial Europeo (ESF) predoctoral fellowship. AUTHOR’s institution issupported by the Programa de Apoyo a Planes Estratégicos de Investigaciónde Estructuras de Investigación de Excelencia co-financed by the Castilla–León autonomous government and the European Regional DevelopmentFund (CLC–2017–01).

Authors’ contributionsManuscript design: SB. Literature search and manuscript drafting: PN, BMLand SB. Manuscript editing, figure design and review: SB. The author(s) readand approved the final manuscript.

Author informationPN’s, BML’s affilition is Centro de Investigación del Cáncer and Instituto deBiología Molecular y Celular del Cáncer, Consejo Superior de InvestigacionesCientíficas (CSIC) - University of Salamanca, 37007 Salamanca, Spain. SB’saffiliations are Centro de Investigación del Cáncer and Instituto de Biología

Molecular y Celular del Cáncer, Consejo Superior de InvestigacionesCientíficas (CSIC) - University of Salamanca, 37007 Salamanca, Spain andInstituto de Investigación Biomédica de Salamanca (IBSAL), HospitalUniversitario de Salamanca, 37007 Salamanca, Spain.

FundingThis work was supported by Spanish Ministry of Economy and Innovation(MINECO), Ministry of Science and Innovation and Agencia Estatal deInvestigación (AEI) and co-financed by the European Development RegionalFund (FEDER) under grant no. SAF2016–78667-R (AEI/FEDER, UE) andPID2019-111692RB-I00. In addition we acknowledge funding from TheScientific Foundation AECC under grant no. LABAE19040BLAN. BML wassupported by Junta de Castilla y León-Fondo Social Europeo (ESF)predoctoral fellowship. AUTHOR’s institution is supported by the Programade Apoyo a Planes Estratégicos de Investigación de Estructuras deInvestigación de Excelencia co-financed by the Castilla–León autonomousgovernment and the European Regional Development Fund (CLC–2017–01).

Availability of data and materialsSupporting data will be available upon request to corresponding author.

Ethics approval and consent to participateNot applicable.

Consent for publicationAll authors read and give final approval of the version to be submitted andany revised version.

Competing interestsThe authors declare that they have no competing interests.

Received: 20 June 2020 Accepted: 24 September 2020

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